Polyhydroxyalkanoate biopolymer compositions

ABSTRACT

Several novel PHA polymer compositions produced using biological systems include monomers such as 3-hydroxybutyrate, 3-hydroxypropionate, 2-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxybutyrate, 4-hydroxyvalerate and 5-hydroxyvalerate. These PHA compositions can readily be extended to incorporate additional monomers including, for example, 3-hydroxyhexanoate, 4-hydroxyhexanoate, 6-hydroxyhexanoate or other longer chain 3-hydroxyacids containing seven or more carbons. This can be accomplished by taking natural PHA producers and mutating through chemical or transposon mutagenesis to delete or inactivate genes encoding undesirable activities. Alternatively, the strains can be genetically engineered to express only those enzymes required for the production of the desired polymer composition. Methods for genetically engineering PHA producing microbes are widely known in the art (Huisman and Madison, 1998, Microbiology and Molecular Biology Reviews, 63: 21-53). These polymers have a variety of uses in medical, industrial and other commercial areas.

[0001] This application claims priority to U.S. Ser. No. 60/086,396filed May 22, 1998.

BACKGROUND TO THE INVENTION

[0002] Numerous microorganisms have the ability to accumulateintracellular reserves of PHA polymers. Poly [(R)-3-hydroxyalkanoates](PHAs) are biodegradable and biocompatible thermoplastic materials,produced from renewable resources, with a broad range of industrial andbiomedical applications (Williams and Peoples, 1996, CHEMTECH 26,38-44). Around 100 different monomers have been incorporated into PHApolymers, as reported in the literature (Steinbüchel and Valentin, 1995,FEMS Microbiol. Lett. 128; 219-228) and the biology and genetics oftheir metabolism has recently been reviewed (Huisman and Madison, 1998,Microbiology and Molecular Biology Reviews, 63: 21-53).

[0003] To date, PHAs have seen limited commercial availability, withonly the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)being available in development quantities This copolymer has beenproduced by fermentation of the bacterium Ralsionia eutropha.Fermentation and recovery processes for other PHA types have also beendeveloped using a range of bacteria including Azotobacter, Alcaligeneslatus, Comamonas testosterone and genetically engineered E. coli andKlebsiella and have recently been reviewed (Braunegg et al., 1998,Journal of Biotechnology 65: 127-161; Choi and Lee, 1999, Appl.Microbiol. Biotechnol. 51: 13-21). More traditional polymer synthesisapproaches have also been examined, including direct condensation andring-opening polymerization of the corresponding lactones (Jesudason andMarchessault, 1994, Macromolecules 27: 2595-2602).

[0004] Synthesis of Pi IA polymers containing the monomer4-hydroxybutyrate (PHB4HB, Doi, Y 1995, Macromol. Symp. 98, 585-599) or4-hydroxyvalerate and 4-hydroxyhexanoate containing PHA polyesters havebeen described (Valentin et al., 1992, Appl. Microbiol. Biotechnol. 36,507-514 and Valentin et al., 1994, Appl. Microbiol. Biotechnol. 40,710-716). These polyesters have been manufactured using methods similarto that originally described for PHBV in which the microorganisms arefed a relatively expensive non-carbohydrate feedstock in order to forcethe incorporation of the monomer into the PHA polyester. The PHB4HBcopolymers can be produced with a range of monomer compositions whichagain provides a range of polymer (Saito, Y, Nakamura, S., Hiramitsu, M.and Doi,, Y., 1996, Polym. Int. 39: 169).

[0005] PHA copolymers of 3-hydroxybutyrate-co-3-hydroxypropionate havealso been described (Shimamura et. al., 1994, Macromolecules 27:4429-4435, Cao et. al., 1997, Macromol. Chem. Phys. 198: 3539-3557). Thehighest level of 3-hydroxypropionate incorporated into these copolymers88 mol % (Shimamura et. al., 1994, Macromolecules 27: 4429-4435).

[0006] PHA terpolymers containing 4-hydroxyvalerate have been producedby feeding a genetically engineered Pseudomonas putida strain on4-hydroxyvalerate or levulinic acid which resulted in a three componentPHA, Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-4-hydroxyvalerate)(Valentin et. al., 1992, Appl. Microbiol. Biotechnol. 36: 507-514;Steinbüchel and Gorenflo, 1997, Macromol. Symp. 123: 61-66). It isdesirable to develop biological systems to produce two componentpolymers comprising 4-hydroxyvalerate or poly(4-hydroxyvalerate)homopolymer. The results of Steinbüchel and Gorenflo (1997, Macromol.Symp. 123: 61-66) indicate that Pseudomonas putida has the ability toconvert levulinic acid to 4-hydroxyvalerate.

[0007] Hein et al. (1997) attempted to synthesize poly-4HV usingtransgenic Escherichia coli strain XL1-Blue but were unsuccessful. Thesecells carried a plasmid which permitted expression of the A. eutrophusPHA synthase and the Clostridium kluyveri 4-hydroxybutyryl-CoAtransferase genes. When the transgenic E. coli were fed 4HV,□-valerolactone, or levulinic acid, they produced only a small amount ofPHB homopolymer.

[0008] It is clearly desirable for industrial reasons to be able toproduce a range of defined PHA homopolymer, copolyer and terpolymercompositions. To accomplish this, it is desirable to be able to controlthe availability of the individual enzymes in the corresponding PHAbiosynthetic pathways.

[0009] It is therefore an object of the present invention to provide arange of defined PHA homopolymer, copolyer and terpolymer compositions.

[0010] It is another object of the present invention to provide a methodand matierlas to control the availability of the individual enzymes inthe corresponding PHA biosynthetic pathways.

SUMMARY OF THE INVENTION

[0011] Several novel PHA polymer compositions produced using biologicalsystems include monomers such as 3-hydroxybutyrate, 3-hydroxypropionate,2-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxybutyrate,4-hydroxyvalerate and 5-hydroxyvalerate. These PHA compositions canreadily be extended to incorporate additional monomers including, forexample, 3-hydroxyhexanoate, 4-hydroxyhexanoate, 6-hydroxyhexanoate orother longer chain 3-hydroxyacids containing seven or more carbons. Thiscan be accomplished by taking natural PHA producers and mutating throughchemical or transposon mutagenesis to delete or inactivate genesencoding undesirable activities. Alternatively, the strains can begenetically engineered to express only those enzymes required for theproduction of the desired polymer composition. Methods for geneticallyengineering PHA producing microbes are widely known in the art (Huismanand Madison, 1998, Microbiology and Molecular Biology Reviews, 63:21-53). These polymers have a variety of uses in medical, industrial andother commercial areas.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a schematic of the pathway from levulinic acid topoly-4-hydroxyvalerate.

[0013]FIG. 2 is a schematic of a construct of plasmid pFS16, whichincludes the lacI (inducer) gene, ampicillin resistance gene, and hbcTgene.

[0014]FIG. 3 is a schematic of a construct of plasmid pFS30, whichincludes the lacI (inducer) gene, ampicillin resistance gene,polyhydroxyalkanoate polymerase (phaC) gene, and hbcT gene.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Several novel PHA polymer compositions have been produced usingbiological systems to incorporate monomers such as 3-hydroxybutyrate,3-hydroxypropionate, 2-hydroxybutyrate, 3-hydroxyvalerate,4-hydroxybutyrate, 4-hydroxyvalerate and 5-hydroxyvalerate. These PHAcompositions can readily be extended to incorporate additional monomersincluding, for example, 3-hydroxyhexanoate, 4-hydroxyhexanoate,6-hydroxyhexanoate or other longer chain 3-hydroxyacids containing sevenor more carbons. Techniques and procedures to engineer transgenicorganisms that synthesize PHAs containing one or more of these monomerseither as sole constituent or as co-monomer have been developed. Inthese systems the transgenic organism is either a bacterium eg.Escherichia coli, K. pneumoniae, Ralstonia eutropha (formerlyAlcaligenes eutrophus), Alcaligenes latus or other microorganisms ableto synthesize PHAs, or a higher plant or plant component, such as theseed of an oil crop (Brassica, sunflower, soybean, corn, safflower,flax, palm or coconut or starch accumulating plants (potato, tapioca,cassava).

[0016] It is crucial for efficient PHA synthesis in recombinant E. colistrains that the expression of all the genes involved in the pathway beadequate. To this end, the genes of interest can be expressed fromextrachromosomal DNA molecules such as plasmids, which intrinsicallyresults in a copy number effect and consequently high expression levels,or, more preferably, they can be expressed from the chromosome. Forlarge scale fermentations of commodity type products it is generallyknown that plasmid-based systems are unsatisfactory due to the extraburden of maintaining the plasmids and the problems of stableexpression. These drawbacks can be overcome using chromosomally encodedenzymes by improving the transcriptional and translational signalspreceding the gene of interest such that expression is sufficient andstable.

[0017] The biological systems must express one or more enzymes asrequired to convert the monomers into polymers. Suitable substratesinclude 3-hydroxybutyrate, 3-hydroxypropionate, 2-hydroxybutyrate,3-hydroxyvalerate, 4-hydroxybutyrate, 4-hydroxyvalerate,5-hydroxyvalerate, 3-hydroxyhexanoate, 4-hydroxyhexanoate,6-hydroxyhexanoate and other longer chain 3-hydroxyacids containingseven or more carbons. These enzymes include polyhydroxyalkanoatesynthase, acyl-CoA transferase and hydroxyacyl CoA transferase, andhydroxyacyl CoA synthetase. These enzymes can be used with thesesubstrates to produce in a biological system such as bacteria, yeast,fungi, or plants, polymer such aspoly(3-hydroxybutyrate-co-4-hydroxyvalerate), poly(4-hydroxyvalerate),poly(3-hydroxypropionate-co-5-hydroxyvalerate), poly(2-hydroxybutyrate),poly(2-hydroxybutyrate-co-3-hydroxybutyrate), andpoly(3-hydroxypropionate).

[0018] Genes encoding the required enzymes can be acquired from multiplesources. U.S. Pat. Nos. 5,798,235 and 5,534,432 to Peoples, et al.,describe polyhydroxyalkanoate synthetase, reductase and thiolase. A4-hydroxybutyryl CoA transferase gene from C. aminobutyricum isdescribed by Willadsen and Buckel, FEMS Microbiol. Lett. (1990) 70:187-192) or from C. kluyveri is described by Söhling and Gottschalk,1996, J. Bacteriol. 178, 871-880). An acyl coenzyme A synthetase fromNeurospora crassa is described by Hii and Courtright, J. Bacteriol.1982.150(2), 981-983. A hydroxyacyl transferase from Clostridium isdescribed by Hofmeister and Bucker, Eur. J. Biochem. 1992, 206(2),547-552.

[0019] It is important for efficient PHA production that strains do notlose the capability to synthesize the biopolymer for the duration of theinoculum train and the production run. Loss of any of the phi genesresults in loss of product. Both are undesirable and stable propagationof the strain is therefore required. Merely integrating the geneencoding the transferase or synthase may not result in significantpolymer production. Enzyme expression can be enhanced through alterationof the promoter region or mutagenesis or other known techniques,followed by screening for polymer production. Growth and morphology ofthese recombinant PHA producers is not compromised by the presence ofpha genes on the chromosome.

[0020] The present invention will be further understood by reference tothe following non-limiting examples.

EXAMPLE 1 Poly(3HB-co-4HV) from 4-hydroxyvalerate and Glucose in E. coli

[0021] Construction of pFS 16.

[0022] The plasmid pTrcN is a derivative of pTrc99a (Pharmacia; Uppsala,Sweden); the modification that distinguishes pTrcN is the removal of theNcoI restriction site by digestion with NcoI, treatment with T4 DNApolymerase, and self-ligation. The orfZ gene encoding the4-hydroxybutyryl-CoA transferase from Clostridium kluyveri was amplifiedusing the polymerase chain reaction (PCR) and a kit from Perkin Elmer(Foster City, Calif.) using plasmid pCK3 (Söhling and Gottschalk, 1996,J. Bacteriol. 178: 871-880) as the target DNA and the followingoligonucleotide primers:5′-TCCCCTAGGATTCAGGAGGTTTTTATGGAGTGGGAAGAGATATATAAAG-3′ (orfZ 5′AvrII)5′-CCTTAAGTCGACAAATTCTAAAATCTCTTTTTAAATTC-3′ (orfZ 3′SalI)

[0023] The resulting PCR product was digested with AvrII and SalI andligated to pTrcN that had been digested with XbaI (which is compatiblewith AvrII) and SalI to form plasmid pFS16 such that the4-hydroxybutyryl-CoA transferase can be expressed from the IPTG(isopropyl-β-D-glucopyranoside)—inducible trcpromoter.

[0024] Construction of pFS30.

[0025] The plasmid pFS30 was derived from pFS16 by adding the Ralstoniaeutropha PHA synthase (phaC) gene (Peoples and Sinskey, 1989. J. Biol.Chem. 264:15298-15303) which had been modified by the addition of astrong E. coli ribosome binding site as described by (Gerngross et al.,1994. Biochemistry 33: 93 11-9320). The plasmid pAeT414 was digestedwith XmaI and StuI so that the R. eutropha promoter and the structuralphaC gene were present on one fragment. pFS16 was cut with BamHI,treated with T4 DNA polymerase to create blunt ends, then digested withXmaI. The two DNA fragments thus obtained were ligated together to formpFS30. In this construct the PHB synthase and 4-hydroxybutyryl-CoAtransferase are expressed from the A. eutrophus phbC promoter (Peoplesand Sinskey, 1989. J. Biol. Chem. 264:15298-15303). Other suitableplasmids expressing PHB synthase and 4-hydroxybutyryl-CoA transferasehave been described (Hein et. al., 1997, FEMS Microbiol. Lett. 153:411-418; Valentin and Dennis, 1997, J. Biotechnol. 58 :33-38).

[0026]E. coli MBX769 has a PHA synthase integrated into its chromosome.This strain is capable of synthesizing poly(3-hydroxybutyrate) (PHB)from glucose with no extrachromosomal genes present. MBX769 is alsodeficient in fadR, the repressor of the fatty-acid-degradation pathwayand effector of many other cellular functions, it is deficient in rpoS,a regulator of stationary-phase gene expression, and it is deficient inatoA, one subunit of the acetoacetyl-CoA transferase. MBX769 alsoexpresses atoC, a positive regulator of the acetoacetate system,constitutively.

[0027]E. coli MBX769 carrying the plasmid pFS16 (FIG. 2), whichpermitted the expression of the Clostridium kluyveri4-hydroxybutyryl-CoA transferase, was precultured at 37° C. in 100 mL ofLB medium containing 100 μg/mL sodium ampicillin in a 250-mL Erlenmeyerflask with shaking at 200 rpm. The cells were centrifuged at 5000 g for10 minutes to remove them from the LB medium after 16 hours, and theywere resuspended in 100 mL of a medium containing, per liter: 4.1 or12.4 g sodium 4-hydroxyvalerate (4HV); 5 g/L sodium 4-hydroxybutyrate(4HB); 2 g glucose; 2.5 g LB broth powder (Difco; Detroit, Mich.); 50mmol potassium phosphate, pH 7; 100 μg/mL sodium ampicillin; and 0.1mmol isopropyl-β-D-thiogalactopyranoside (IPTG). The sodium4-hydroxyvalerate was obtained by saponification of γ-valerolactone in asolution of sodium hydroxide. The cells were incubated in this mediumfor 3 days with shaking at 200 rpm at 32° C. in the same flask in whichthey had been precultured. When 4.1 g/L sodium 4-hydroxyvalerate waspresent initially, the cells accumulated a polymer to 52.6% of the drycell weight that consisted of 63.4% 3HB units and 36.6% 4HB units but no4HV units.

[0028] When 12.4 g/L sodium 4HV was present initially, the cellsaccumulated a polymer to 45.9% of the dry cell weight that consisted of95.5% 3HB units and 4.5% 4HV units but no detectable 4HB units. Theidentity of the PHB-co-4HV polymer was verified by nuclear magneticresonance (NMR) analysis of the solid product obtained by chloroformextraction of whole cells followed by filtration, ethanol precipitationof the polymer from the filtrate, and washing of the polymer with water.It was also verified by gas chromatographic (GC) analysis, which wascarried out as follows. Extracted polymer (1-20 mg) or lyophilized wholecells (15-50 mg) were incubated in 3 mL of a propanolysis solutionconsisting of 50% 1,2-dichloroethane, 40% 1-propanol, and 10%concentrated hydrochloric acid at 100° C. for 5 hours. The water-solublecomponents of the resulting mixture were removed by extraction with 3 mLwater. The organic phase (1 μL at a split ratio of 1:50 at an overallflow rate of 2 mL/min) was analyzed on an SPB-1 fused silica capillaryGC column (30 m; 0.32 mm ID; 0.25 μm film; Supelco; Bellefonte, Pa.)with the following temperature profile: 80° C., 2 min; 10 C° per min to250° C.; 250° C., 2 min. The standard used to test for the presence of4HV units in the polymer was γ-valerolactone, which, like4-hydroxyvaleric acid, forms propyl 4-hydroxyvalerate upon propanolysis.The standard used to test for 3HB units in the polymer was PHB.

EXAMPLE 2 Poly(4HV) from 4-hydroxyvalerate in E. coli

[0029]Escherichia coli MBX1177 is not capable of synthesizingpoly(3-hydroxybutyrate) (PHB) from glucose. MBX1177 is a spontaneousmutant of strain DH5□ that is able to use 4-hydroxybutyric acid as acarbon source. MBX1177 carrying the plasmid pFS30 (FIG. 2), whichpermitted the expression of the Clostridium kluyveri 4HB-CoA transferaseand the Ralstonia eutropha PHA synthase, was precultured at 37° C. in100 mL of LB medium containing 100 μg/mL sodium ampicillin.

[0030] The cells were centrifuged at 5000 g for 10 minutes to removethem from the LB medium after 16 hours, and they were resuspended in 100mL of a medium containing, per liter: 5 g sodium 4-hydroxyvalerate(4HV); 2 g glucose; 2.5 g LB broth powder; 100 mmol potassium phosphate,pH 7; 100 μg/mL sodium ampicillin, and 0.1 mmol IPTG. The cells wereincubated in this medium for 3 days with shaking at 200 rpm at 30° C. inthe same flask in which they had been precultured.

[0031] The cells accumulated a polymer to 0.25% of the dry cell weightthat consisted of 100% 4HV units. The identity of the poly(4HV) polymerwas verified by GC analysis of whole cells that had been washed withwater and propanolyzed in a mixture of 50% 1,2-dichloroethane, 40%I-propanol, and 10% concentrated hydrochloric acid at 100° C. for 5hours, with γ-valerolactone as the standard.

EXAMPLE 3 Poly(3HB-co-2HB) from 2-hydroxybutyrate and Glucose in E. coli

[0032]E. coil MBX769 carrying the plasmid pFS16 was precultured at 37°C. in 100 mL of LB medium containing 100 μg/mL sodium ampicillin in a250-mL Erlenmeyer flask with shaking at 200 rpm. The cells werecentrifuged at 5000 g for 10 minutes to remove them from the LB mediumafter 16 hours, and they were resuspended in 100 mL of a mediumcontaining, per liter: 5 g sodium 2-hydroxybutyrate (2HB); 2 g glucose;2.5 g LB broth powder; 50 mmol potassium phosphate, pH 7; 100 μg/mLsodium ampicillin; and 0.1 mmol IPTG. The cells were incubated in thismedium for 3 days with shaking at 150 rpm at 33° C. in the same flask inwhich they had been precultured. The cells accumulated a polymer to19.0% of the dry cell weight that consisted of 99.7% 3HB units and 0.3%2HB units. The identity of the poly(3HB-co-2HB) polymer was verified byGC analysis of the solid product obtained by chloroform extraction ofwhole cells followed by filtration) ethanol precipitation of the polymerfrom the filtrate, and washing of the polymer with water. It was alsoverified by GC analysis of whole cells that had been washed with waterand propanolyzed in a mixture of 50% 1,2-dichloroethane, 40% 1-propanol,and 10% concentrated hydrochloric acid at 100° C. for 5 hours, with PHBand sodium 2-hydroxybutyrate as the standards.

EXAMPLE 4 Poly(2HB) from 2-hydroxybutyrate in E. coli

[0033]Escherichia coli MBX184 is not capable of synthesizingpoly(3-hydroxybutyrate) (PHB) from glucose. MBX184 is deficient in fadRand expresses atoC constitutively.

[0034] MBX184 carrying the plasmid pFS30 was precultured at 37° C. in100 mL of LB medium containing 100 μg/mL sodium ampicillin. The cellswere centrifuged at 5000 g for 10 minutes to remove them from the LBmedium after 16 hours, and they were resuspended in 100 mL of a mediumcontaining, per liter: 5 g sodium 2-hydroxybutyrate (2HB); 2 g glucose,2.5 g LB broth powder, 50 mmol potassium phosphate, pH 7; 100 μg/mLsodium ampicillin; and 0.1 mmol IPTG. The cells were incubated in thismedium for 3 days with shaking at 150 rpm at 33° C. in the same flask inwhich they had been precultured.

[0035] The cells accumulated a polymer to 1.0% of the dry cell weightthat consisted of 100% 2HB units. The identity of the poly(2HB) polymerwas verified by GC analysis of whole cells that had been washed withwater and propanolyzed in a mixture of 50% 1,2-dichloroethane, 40%1-propanol, and 10% concentrated hydrochloric acid at 100° C. for 5hours, with sodium 2-hydroxybutyrate as the standard.

EXAMPLE 5 Poly-3HP and poly-3HP-co-5HV from 1,3-propanediol and from1,5-pentanediol

[0036]Escherichia coli MBX184 carrying the plasmid pFS30 was preculturedat 37° C. in 100 mL of LB medium containing 100 μg/mL sodium ampicillin.The cells were centrifuged at 5000 g for 10 minutes to remove them fromthe LB medium after 16 hours, and they were resuspended in 100 mL of amedium containing, per liter: 10 g 1,3-propanediol (1,3-PD) or1,5-pentanediol (1,5-PD); 2 g glucose; 2.5 g LB broth powder; 50 mmolpotassium phosphate, pH 7; 100 μg/mL sodium ampicillin; and 0.1 mmolIPTG. The cells were incubated in this medium for 3 days with shaking at200 rpm at 30° C. in the same flask in which they had been precultured.When the diol substrate was 1,3-PD, the cells accumulated a polymer to7.0% of the dry cell weight that consisted entirely of 3HP units. Whenthe substrate was 1,5-PD, the cells accumulated a polymer to 22.1% ofthe dry cell weight that consisted of greater than 90%3-hydroxypropionate units and less than 10% 5-hydroxyvalerate units. Theidentity of the poly(3-hydroxypropionate) polymer was verified by NMRanalysis of the solid product obtained by sodium hypochlorite extractionof whole cells followed by centrifugation and washing of the polymerwith water. The identity of both polymers was verified by CC analysis ofsodium hypochlorite-extracted polymer that was propanolyzed in a mixtureof 50% 1,2-dichloroethane, 40% 1-propanol, and 10% concentratedhydrochloric acid at 100° C. for 5 hours, with , β-propiolactone andδ-valerolactone as the standards.

EXAMPLE 6 Poly-5HV from 5-hydroxyvaleric Acid

[0037]Escherichia coli MBX1177 carrying the plasmid pFS30 wasprecultured at 37° C. in 50 mL of LB medium containing 100 μg/mL sodiumampicillin. The cells were centrifuged at 5000 g for 10 minutes toremove them from the LB medium after 8 hours, and they were resuspendedin 100 mL of a medium containing, per liter: 10 g sodium5-hydroxyvalerate (5HV), 5 g glucose, 2.5 g LB broth powder; 50 mmolpotassium phosphate, pH 7, 100 μg/mL sodium ampicillin; and 0.1 mmolIPTG. The sodium 5HV was obtained by saponification of d-valerolactone.The cells were incubated in this medium for 3 days with shaking at 200rpm at 30° C. in the same flask in which they had been precultured. GCanalysis was conducted with lyophilized whole cells that werebutanolyzed in a mixture of 90% 1-butanol and 10% concentratedhydrochloric acid at 110° C. for 5 hours; the standard was sodium5-hydroxyvalerate. This analysis showed that the cells had accumulatedpoly(5HV) to 13.9% of the dry cell weight. The identity of thepoly(5-hydroxyvalerate) polymer was verified by NMR analysis of thesolid product obtained by 1,2-dichloroethane extraction of whole cellsfollowed by centrifugation and washing of the polymer with water.

[0038] Modifications and variations are intended to come within thescope of the appended claims.

1 2 1 49 DNA Artificial Sequence Description of Artificial SequencePrimer- orfZ 5′ AvrII 1 tcccctagga ttcaggaggt ttttatggag tgggaagagatatataaag 49 2 38 DNA Artificial Sequence Description of ArtificialSequence Primer- orfZ 3′ SalI 2 ccttaagtcg acaaattcta aaatctctttttaaattc 38

We claim:
 1. A polymer produced by providing one or more substratesselected from the group consisting of 3-hydroxybutyrate,3-hydroxypropionate, 2-hydroxybutyrate, 3-hydroxyvalerate,4-hydroxybutyrate, 4-hydroxyvalerate, 5-hydroxyvalerate,3-hydroxyhexanoate, 4-hydroxyhexanoate,. 6-hydroxyhexanoate and otherlonger chain 3-hydroxyacids containing seven or more carbons, whereinthe biological system expresses enzymes selected from the groupconsisting polyhydroxyalkanoate synthase, acyl-CoA transferase,hydroxyacyl CoA transferase, and hydroxyacyl CoA synthetase such thatthe polymers accumulate.
 2. The polymer of claim 1 selected from thegroup consisting of poly(3-hydroxybutyrate-co-4-hydroxyvalerate),poly(4-hydroxyvalerate), poly(3-hydroxypropionate-co-5-hydroxyvalerate),poly(2-hydroxybutyrate), poly(2-hydroxybutyrate-co-3-hydroxybutyrate),poly(3-hydroxypropionate), produced in a biological system selected fromthe group comprising bacteria, yeasts, fungi and plants, wherein thebiological system expresses enzymes selected from the group consistingpolyhydroxyalkanoate synthase, acyl-CoA transferase and hydroxyacyl CoAtransferase, and hydroxyacyl CoA synthetase such that the polymersaccumulate in the presence of appropriate substrates.
 3. The polymer ofclaim 1 wherein the polymer ispoly(3-hydroxybutyrate-co-4-hydroxyvalerate),
 4. The polymer of claim 1wherein the polymer is poly(4-hydroxyvalerate).
 5. The polymer of claim1 wherein the polymer is poly(3-hydroxypropionate-co-5-hydroxyvalerate).6. The polymer of claim 1 wherein the polymer ispoly(3-hydroxypropionate).
 7. A polyhydroxyalkanoate polymer comprising2-hydroxybutyrate as a comonomer, wherein the polymer is produced in abiological system selected from the group comprising bacteria, yeasts,fungi group consisting polyhydroxyalkanoate synthase, acyl-CoAtransferase, hydroxyacyl CoA transferase) and hydroxyacyl CoA synthetasesuch that the polymers accumulate in the presence of appropriatesubstrates.
 8. The polymer of claim 7 wherein the polymer ispoly(2-hydroxybutyrate).
 9. The polymer of claim 7 wherein the polymeris poly(2-hydroxybutyrate-co-3-hydroxybutyrate).
 10. A method for makingpolymers in a biological system comprising providing one, or moresubstrates selected from the group consisting of 3-hydroxybutyrate,3-hydroxypropionate, 2-hydroxybutyrate, 3-hydroxyvalerate,4-hydroxybutyrate, 4-hydroxyvalerate, 5-hydroxyvalerate,3-hydroxyhexanoate, 4-hydroxyhexanoate, 6-hydroxyhexanoate and otherlonger chain 3-hydroxyacids containing seven or more carbons, whereinthe biological system expresses enzymes selected from the groupconsisting polyhydroxyalkanoate synthase, acyl-CoA transferase,hydroxyacyl CoA transferase, and hydroxyacyl CoA synthetase such thatthe polymers accumulate.
 11. The method of claim 10 wherein theorganisms express one or more heterologous genes encoding the enzymes.12. The method of claim 10 for making a copolymer of 3-hydroxybutyrateand 4-hydroxybutyrate comprising incubating equimolar amounts of(R)-3-hydroxybutyrate and 4-hydroxybutyrate with 4-hydroxybutyrate CoAtransferase.